Aus dem Pathologischen Institut der Ludwig-Maximilians-Universität München
Direktor: Prof. Dr. med. Thomas Kirchner
in der Arbeitsgruppe Experimentelle und Molekulare Pathologie
Leiter: Prof. Dr. rer. nat. Heiko Hermeking
Genetic analysis of Ap4 in the Apc
Minmouse model
Dissertation zum Erwerb des Doktorgrades der
Naturwissenschaften (Dr. rer. nat.) an der Medizinischen Fakultät
der Ludwig-Maximilians-Universität München
vorgelegt von
Stephanie Jaeckel
aus Flensburg
Gedruckt mit der Genehmigung der Medizinischen Fakultät
der Ludwig-Maximilians-Universität München
Erstgutachter: Prof. Dr. rer. nat. Heiko Hermeking
Zweitgutachter: Priv. Doz. Dr. rer. nat. Andreas Herbst
Dekan: Prof. Dr. med. dent. Reinhard Hickel
I
Eidesstattliche Versicherung
Stephanie Jaeckel
Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema
“Genetic analysis of Ap4 in the ApcMin mouse model”
selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe.
Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde.
Publications
II
Publications
Parts of this thesis have been published in the article:
Stephanie Jaeckel, Markus Kaller, Rene Jackstadt, Ursula Götz, Susanna Müller, Sophie Boos, David Horst, Peter Jung and Heiko Hermeking. Ap4 is rate limiting for intestinal tumor formation by controlling the homeostasis of intestinal stem cells. Nature Communications; 2018; 9: 3573.
Abbreviations III Abbreviations ABC AEC AP4 APC APCMin APS bHLH-LZ cDNA ChIP ChIP-seq CMS CRC Cre CSC Avidin-Biotin complex 3-Amino-9-ethylcarbazole
Activating enhancer binding protein 4 Adenomatous polyposis coli
APCMultiple intestinal neoplasia
Ammonium peroxodisulfate
basic-Helix-loop-helix leucine zipper complementary DNA
Chromatin immuno-precipitation ChIP-sequencing
Consensus molecular subtypes Colorectal cancer
Cyclization recombination or “causes recombination” Cancer stem cell
CSL CTTNB1 DAB DAPI
CBF1/suppressor of Hairless/LAG-1 Catenin beta 1, gene encoding β-catenin 3,3'-diaminobenzidine 2-(4-Amidinophenyl)-6-indolecarbamidine-dihydrochloride DBZ DCS DIG DMEM DMSO
Dibenzazepine (inhibitor of γ-secretase) Deep crypt secretory cells
Digoxigenin
Dulbecco`s modified Eagles medium Dimethyl-sulfoxide DNA DOX dNTPs E-Box E.coli ECL EDTA EGF eGFP EM EMT EMT-TF ER ERT2 ES cells FAP FBS FFPE FITC fl flp frt Deoxyribonucleic acid Doxycycline Deoxynucleotides triphosphate Enhancer box Escherichia coli Enhanced chemiluminescence Ethylene-diamine-tetra-acetic acid Epidermal growth factor
enhanced Green fluorescent protein Electron microscopy
Epithelial-mesenchymal transition EMT-inducing transcription factor Endoplasmatic reticulum
Estrogen receptor tamoxifen inducible 2 Embryonic stem cells
Familial adenomatous polyposis Fetal bovine serum
Formalin-fixed, paraffin-embedded Flourescein
Floxed, flanked by loxP Flippase
Abbreviations IV GSEA HBSS HE HES HRP IEC IF IgG IHC ISC ISH IVC Kbp KD=kDa KEGG Ko LGR5 loxP LRC LSM MEF MET mRNA MSigDB NEO NGS NICD 4-OHT OLFM4 ORF PAGE PAS PBS PCR PFA P/S qPCR RBP-Jκ RNA RNA-Seq Rpkm RSEM RSPO1 SD SDS siRNA SMOC2 TAM
Gene set enrichment analysis Hank’s balanced salt solution Hematoxylin and eosin
Hairy and enhancer of split Horse-radish-peroxidase Intestinal epithelial cell Immunofluorescence Immunoglobulin G Immunohistochemistry Intestinal stem cell
In situ hybridization
Individually ventilated cages Kilo base pair
Kilo dalton
Kyoto Encyclopedia of Genes and Genomes Knockout
Leu-rich repeat-containing G protein-coupled Receptor 5 Locus of X-over P1
Label retaining cell
Laser scanning microscopy Mouse embryonic fibroblast Mesenchymal-epithelial transition Messenger RNA
Molecular signatures database Neomycin
Next-generation sequencing Notch intracellular domain 4-hydroxytamoxifen
Olfactomedin 4 Open reading frame
Polyacrylamide gel electrophoresis Periodic acid-Schiff
Phosphate buffered saline Polymerase chain reaction Paraformaldehyde
Penicillin Streptomycin Quantitative real-time PCR
Recombination signal binding protein for immunoglobulin kappa J region
Ribonucleic acid RNA-Sequencing
Reads per kilobase million
RNA-Seq by expectation maximization R-spondin-1
Standard deviation Sodium dodecyl sulfate Small interfering RNA
SPARC-related modular calcium-binding protein 2 Tamoxifen
Abbreviations V TA unit TCF TCGA TEMED TFAP4 TMA TSS Transient-amplifying unit T-cell factor
The cancer genome atlas
Tetramethylethylendiamin,1,2-bis (dimethylamino) - ethan Transcription factor AP4
Tissue microarray Transcriptional start site Vil-Cre
VSV WB
Villin-Cre
Vesicular stomatitis virus (tag) Western blot analysis
WNT Wingless/Int-1
Table of contents VI Table of contents Eidesstattliche Versicherung ... I Publications ... II Abbreviations ... III 1. Introduction ... 1 1.1 Cancer incidence ... 1
1.2 The hallmarks of cancer ... 2
1.3 The genetics of colorectal cancer ... 3
1.4 Canonical Wnt-signaling in colorectal cancer ... 4
1.5 The Notch signaling in colorectal cancer ... 5
1.6 The transcription factor AP4 ... 8
1.7 The role of AP4 in colorectal cancer... 10
1.8 The homeostasis of the intestinal epithelium ... 11
1.9 The role of the Wnt and Notch pathway in intestinal homeostasis ... 13
2. Aims of the study ... 16
3. Materials ... 17
3.1 Chemicals and reagents ... 17
3.2 Enzymes ... 19
3.3 Kits ... 19
DAB+ Substrate Chromogen System ... 19
3.4 Antibodies ... 20
3.4.1 Primary antibodies ... 20
3.4.2 Secondary antibodies ... 21
3.5 Buffers and solutions ... 22
3.6 Oligonucleotides ... 26
3.6.1 Oligonucleotides used for genotyping ... 26
3.6.2 Oligonucleotides used for qPCR ... 26
3.6.3 Oligonucleotides used for qCHIP ... 27
3.7 siRNAs ... 29
Table of contents VII 3.9 Mice ... 29 3.10 Cell lines ... 30 3.11 Software ... 30 3.12 Laboratory equipment ... 31 4. Methods ... 32
4.1 Generation and husbandry of mice... 32
4.2 Tissue preparation and adenoma counting ... 32
4.3 HE and PAS/Alcian blue staining... 33
4.4 Immunohistochemistry ... 33
4.5 In situ hybridization ... 34
4.6 Isolation of IECs ... 34
4.7 Crypt isolation and organoid culture ... 34
4.8 Tissue microarrays and IHC (immunohistochemistry) analysis of clinical samples ... 36
4.9 RNA expression profiling by RNA-Seq ... 36
4.10 Bioinformatics analyses of RNA-Seq and ChIP-Seq data ... 36
4.11 In silico analysis of human colorectal patient samples ... 37
4.12 Indirect immunofluorescence detection and confocal laser-scanning microscopy ... 38
4.13 Electron microscopy ... 38
4.14 RNA isolation and quantitative real-time PCR (qPCR) ... 38
4.15 DBZ treatment of mice ... 38
4.16 Cell lines / culture and reagents ... 39
4.17 Chromatin immunoprecipitation (ChIP) assay ... 39
4.18 Generation of cell pools stably expressing conditional alleles ... 40
4.19 Plasmids and RNAi ... 40
4.20 Cell-Based Reporter Assays ... 40
4.21 Western blot analysis ... 41
4.22 Statistical analysis ... 41
4.23 Data availability statement ... 41
Table of contents
VIII
5.1 Role of Ap4 in intestinal adenoma formation ... 42
5.2 mRNA expression profiling of Ap4-deficient adenomas ... 49
5.3 Analysis of tumor organoids from Ap4-deficient ApcMin mice ... 57
5.4 Ap4 regulates the homeostasis of intestinal stem cells... 62
5.5 Analysis of Ap4 function in intestinal organoids ... 73
5.6 Gene expression profiling of Ap4-deficient organoids... 74
5.7 Regulation of the NOTCH pathway by AP4 in human CRC cells ... 80
5.8 Role of AP4 in human CRCs ... 86
6. Discussion ... 90 7. Summary ... 97 8. Zusammenfassung ... 98 9. Acknowledgements ... 99 10. References ... 100 11. Supplements ... 113 11.1 Supplemental Data 1 ... 113 11.2 Supplemental Data 2 ... 128 11.3 Supplemental Data 3 ... 146
Introduction
1
1. Introduction
1.1 Cancer incidence
Today, cancer is the second major reason for global death: 9,6 million people have died of cancer in 2018, while 18,1 million new cases have been registered (Figure 1) (as of 12th of September 2018 at (www.who.int/cancer/PRGlobocanFinal.pdf?ua=1)).
Figure 1 Cancer incidence in 2018. Number of new cases (upper panel) and cancer related deaths (lower panel) in 2018. Colorectal cancer is highlighted. Taken from: http://gco.iarc.fr/today/data/factsheets/cancers/10_8_9-Colorectum-fact-sheet.pdf. As of 12th of September 2018.
Introduction
2 Colorectal cancers (CRCs) constitute about 1.8 million new cases and 10.2% of all new cancer cases in 2018 worldwide, while CRC related deaths include 881.000 cases and 9,2% of all cancer related deaths. CRC is the third most frequent cause of new cancer cases and the second most frequent cause of cancer related deaths worldwide (Figure 1).
1.2 The hallmarks of cancer
Cancer develops after an accumulation of mutations in genes leading to constant activation of cancer promoting oncogenes or inactivation of tumor suppressor genes. While tumor promoting oncogenes only require a single mutation for activation, tumor suppressor genes require two mutations for inactivation - one mutation in both alleles. These mutations are responsible for the six so-called hallmarks of cancer that leads to survival and proliferation of cancer cells followed by metastasis to other tissues and organs in the body: sustaining proliferation, evading growth suppressors, resisting apoptosis, inducing angiogenesis, enabling replicative immortality and activation of invasion and metastasis (Figure 2) (Hanahan and Weinberg, 2000, 2011).
Figure 2 The Hallmarks of Cancer. Schematic representation of the six hallmarks of cancer. Taken from (Hanahan and Weinberg, 2011).
Introduction
3
1.3 The genetics of colorectal cancer
Around 85% of all human colorectal cancers have mutations in the APC (Adenomatous polyposis coli) gene (Kinzler and Vogelstein, 1996), which is true for both sporadic CRC (Payne, 1990) and inherited CRC (Powell et al., 1993). An activation of the WNT (Wingless/Int-1)-signaling pathway by mutations in APC or
CTNNB1 (Catenin Beta 1, gene encoding β-CATENIN) is the initiating event in CRC
(Figure 3). The β-CATENIN/TCF4 (T-cell factor 4) complex induces the expression of a large number of genes, which maintain intestinal stem cells (ISCs) in an undifferentiated state. Uncontrolled proliferation of these cells results in benign adenomas, which progress to malignant intestinal cancer with almost 100% probability (Clevers and Nusse, 2012). During progression, additional mutations occur, as for example the oncogenic activation of K-RAS and the inactivation of the tumor-suppressor gene p53 (Fearon and Vogelstein, 1990; Kinzler and Vogelstein, 1996; Sancho et al., 2004).
Figure 3 Genetic mutations correlated with colorectal tumorigenesis. Mutation in the APC gene initiate the process of colorectal tumorigenesis. Mutations in additional, indicated genes result in further progression. Taken from (Kinzler and Vogelstein, 1996).
The ApcMin (Multiple intestinal neoplasia) mouse model is commonly used for the study of
genes involved in colorectal cancer. These mice harbor a mutation in codon 850 in the
Apc gene leading to a premature stop in translation resulting in an incomplete Apc
protein, which is no longer able to inactivate Wnt signaling. The consequence is the development of about 100 adenomas in the small intestine and few adenomas in the colon which represent the early stage of colorectal cancer (Moser et al., 1990; Su et al., 1992). The human and mouse Apc genes display sequence similarity of around 90% (Su et al., 1992). The ApcMin mouse model and the human inherited human
familial adenomatous polyposis (FAP) syndrome show high genetic and phenotypic similarities. In humans the FAP syndrome leads to 100-1000 benign tumors in the
Introduction
4 colon which ultimately develop into malignant colorectal cancer in all cases. Since the
APC gene is mutated in almost all human colorectal cancers, ApcMin mice represent a
relevant mouse model of colorectal cancer (Su et al., 1992). Due to high tumor burden and a consequently short lifespan of mice with a C57BL/6 background, tumor cells in the adenomas do not accumulate further mutations necessary for progression into adenocarcinomas and metastasis (Halberg et al., 2009). Only in a mixed background causing lower tumor burden and subsequent longer survival time, the adenomas progress into malignant cancer (Halberg et al., 2009). Early detection of CRC is critical for curing cancer and the understanding of the earliest mechanisms in the development of CRC is necessary. The mutation of Apc is modelling the initiation of human CRC, which makes it possible to study the early steps of cancer progression.
1.4 Canonical Wnt-signaling in colorectal cancer
The canonical Wnt signaling pathway is highly conserved between species. In the absence of Wnt ligands, β-catenin is phosphorylated by the destruction complex. The destruction complex consists of Axin (axis inhibition protein), which functions as a scaffold protein, the tumor suppressor protein Apc (adenomatous polyposis coli) as well as the protein kinases Ck1 (Casein kinase 1) and Gsk3 (Glycogen synthase kinase 3), which phosphorylate β-catenin. The phosphorylation of β-catenin leads to its degradation by the β-transducin-repeat-containing protein (βTrCP). In the absence of
β-catenin, Tcf transcription factors bind to Groucho corepressors resulting in repression of transcription of Wnt target genes (Figure 4, left panel). When the Wnt ligand binds to the complex of the receptor Frizzled (Fz) and its coreceptor Lrp (lipoprotein receptor-related protein), Axin as well as Ck1 and Gsk3 are recruited to the Lrp receptor and no destruction complex is formed. β-catenin enters the nucleus, where it displaces the Groucho repressors by binding to Tcf-transcription factor proteins to promote the transcription of Wnt target genes (Figure 4, right panel).
Introduction
5 Figure 4 Canonical Wnt signaling. Molecular processes in the Wnt
signaling pathway are illustrated. See main text for further explanation. Taken from (Clevers, 2006).
1.5 The Notch signaling in colorectal cancer
The Notch signaling pathway consists of four different Notch receptors (Notch1-4) and five Notch ligands from the Dsl (Delta/Serrate/Lag-2) transmembrane protein family, three are delta like (ligand) proteins (Dll1, Dll3 and Dll4) and two are the Jagged proteins (Jag1 and Jag2). Three cleavages (S1-S3) of the native Notch protein occur during maturation and signaling activation (Figure 5). For maturation, the immature Notch receptor is glycosylated by the proteins Pofut1 (protein O-fucosyltransferase 1) and Fringe in the endoplasmic reticulum (ER) which leads to cleavage by a furin-like convertase (S1). After binding of the DSL ligand presented by the signal-sending cell to the Notch receptor at the membrane of the signal-receiving cell, Adam (a disintegrin and metalloprotease pepdidase) cleaves the Notch receptor (S2), after which the Notch extracellular domain is endocytosed by the signal-sending cell. The gamma (γ )-secretase cleaves the intracellular domain of the Notch receptor (S3), which releases the Notch intracellular domain (Nicd) into the cytoplasm. In the nucleus, Nicd forms a transcriptional complex with the DNA binding protein Rbp-Jκ (recombination signal binding protein for immunoglobulin kappa J region), also known as Csl
Introduction
6 (Cbf1/suppressor of hairless/Lag-1), as well as other transcriptional coactivators such as Maml1 (mastermind like transcriptional coactivator 1). This complex mediates target gene regulation (Noah and Shroyer, 2013). A known Notch target gene is Hes1 (hairy and enhancer of split-1), whose gene transcription is activated in response to an active Notch signaling (Fre et al., 2005; Jarriault et al., 1995; Jarriault et al., 1998). For inactivation, Nicd is ubiquitinated by Fbw7 (F-box and WD repeat domain-containing 7). The ubiquitination targets Nicd for degradation (Noah and Shroyer, 2013). Another function of the DSL on the signal-sending cell is to inhibit their own Notch receptor. Furthermore, the DSL ligand undergoes proteolytic cleavage by Adam and γ -secretase, whereafter it binds to Rbp-Jκ (Csl) in combination with corepressors (CoR) to inhibit Notch target gene expression. The lack of Hes1 expression leads to expression of Atoh1 (protein atonal homolog 1), which induces transcription of new DSL ligands (Noah and Shroyer, 2013).
Introduction
7 Figure 5 The Notch signaling pathway. a Schematic representation of the Notch receptors and ligands (Delta/Serrate/Lag-2: DSL). NRR: negative regulatory region, EGF-like repeat: epidermal growth factor-like repeat, LNR module: Lin12-Notch repeat module, RAM domain: RBP-Jkappa-associated module domain, PEST sequence: a peptide sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T), DOS domain: Dll4 delta and Osm‐11 domain, PDZ ligand motif: domain present in Psd-95, Dlg, and Zo-1/2. b Molecular processes in the Notch signaling pathway. See main text for further explanation. ER: endoplasmic reticulum. Taken from (Noah and Shroyer, 2013).
Introduction
8 NOTCH signaling is deregulated in human CRCs and is active during the whole process of tumor formation (Wu et al., 2013). In tumor development, NOTCH1-4 seems to have different functions. Upregulation of NOTCH1 (Chu et al., 2011) and NOTCH3 (Serafin et al., 2011) positively correlates with poor survival in patients with CRC. In addition, ectopic expression of NOTCH1 (Wu et al., 2013; Zhang et al., 2010) or NOTCH3 (Serafin et al., 2011) promotes tumor progression and metastasis. However, NOTCH1 and NOTCH2 negatively correlate with each other and NOTCH2 expression negatively correlates with poor survival in contrast to NOTCH1 (Chu et al., 2011). Ectopic NOTCH4 expression in human CRC cell lines leads to a decreased proliferation rate as well as decreased migration and invasion (Zhang et al., 2018). This means, while NOTCH1 and NOTCH3 support tumorigenesis and metastasis in human CRC, NOTCH2 and NOTCH4 inhibit the same event. However, high expression of both NOTCH1 and NOTCH2 is associated with the poorest survival (Chu et al., 2011). Therefore, an interplay of different NOTCH receptors results in other effects than elevated expression of a single receptor.
Like in humans, Notch signaling plays a role in tumorigenesis in mice, since activated Notch in intestinal epithelial cells of ApcMin mice leads to an increase in intestinal
adenomas and a shortened lifespan (van Es et al., 2005b). Furthermore, there is a link between the cancer initiating Wnt/β-catenin pathway activation and Notch signaling, since a high level of β-catenin activates the transcription of the Notch ligand Jag1 in
ApcMin mice (Rodilla et al., 2009), which could be, at least in part, the cause for
activation of both pathways in intestinal tumorigenesis.
1.6 The transcription factor AP4
The TFAP4/AP4 (transcription factor activating enhancer binding protein 4) protein belongs to the class of basic-helix-loop-helix leucine zipper (bHLH-LZ) transcription factors. AP4 exclusively forms homodimers via interaction of the three protein dimerization sites HLH, LZ1 and LZ2. The AP4 homodimers use the N-terminal basic region to bind to the E-box motif CA(G/C)CTG in the promoter, thereby activating the expression of target genes. For repression of target genes the AP4 homodimer binds to a geminin-SMRT complex to recruit HDAC3 (Histone deacetylase 3) or it binds directly to HDAC1. The AP4 protein contains a highly conserved TIV motif consisting of the amino acids Threonine (T), Isoleucine (I) and Valine (V), but its function is
Introduction
9 unknown (Figure 6) (reviewed in (Jung and Hermeking, 2009)). The gene encoding AP4 was previously identified as a direct transcriptional target of c-MYC (Jung et al., 2008). c-MYC also belongs to the bHLH-LZ transcription factors, but binds to the E-box motif CA(C/T)GTG as a heterodimer with MAX (reviewed in (Jung and Hermeking, 2009)).
Figure 6 Scheme of the structure of the AP4 protein and its molecular interactions. B: basic region, HLH: helix-loop-helix, LZ: leucine zipper, HDAC: histone deacetylase, AA: amino acid position. Taken from (Jung and Hermeking, 2009).
In human CRC cells AP4 directly repress the gene encoding the CDK-inhibitor p21 (encoded by the gene CDKN1A) to block differentiation and to induce proliferation and apoptosis. This result points to an important role of AP4 in mediating the repressing effect of c-MYC on p21 expression. In addition, AP4 is expressed in progenitor/transient amplifying cells in human colonic crypts as well as in colorectal cancer in a pattern similar to c-MYC and in a converse pattern to p21, indicating an identical or at least similar function of AP4 in human tissue as in human cell lines (Jung et al., 2008).
Introduction
10
1.7 The role of AP4 in colorectal cancer
The prototypic oncogene c-MYC is a direct target of the APC/WNT pathway (He et al., 1998) and an essential mediator of tumor formation induced by inactivation of Apc in the intestine of mice (Sansom et al., 2007; Sur et al., 2012). Deletion of one c-Myc allele decreases the tumor load in ApcMin mice (Sur et al., 2012), while a complete loss
of c-Myc prevents adenoma formation caused by homozygous Apc deletion in mice (Sansom et al., 2007). In addition, both human and murine c-MYC are direct targets of the Notch pathway (Palomero et al., 2006; Weng et al., 2006). Therefore, c-MYC may be an important effector of APC/WNT-induced and/or NOTCH-induced tumor formation in the intestine and direct target genes of c-MYC could have an important influence in the development and progression of cancer. High expression of the c-MYC target gene AP4 positively correlated with survival and formation of distant metastases in the liver in two different colorectal cancer patient cohorts (Jackstadt et al., 2013c). Additionally, high expression of AP4 also correlated with poor survival in other tumor entities, such as gastric cancer, non-small cell lung carcinomas and hepatocellular tumors (Gong et al., 2014; Hu et al., 2013; Xinghua et al., 2012). Moreover, a genome-wide analysis of AP4-regulated genes and AP4 DNA binding has been performed in a human colon cancer (CRC) cell line (Jackstadt et al., 2013c). Interestingly, AP4 could be identified as an epithelial-mesenchymal transition inducing transcription factor (EMT-TF), which regulates a large number of genes controlling EMT, stemness and proliferation. AP4 was required for metastases formation by CRC lines in a xenograft mouse model (Jackstadt et al., 2013c). Furthermore, a double negative feedback-loop between AP4 and the tumor-suppressor microRNA-15a/16-1 controls the balance of EMT and MET (mesenchymal-epithelial transition) during metastases formation of human CRCs (Shi et al., 2014). In addition, microRNA-302c directly targets AP4 and represses the transcription of AP4, thereby repressing EMT and metastasis formation of CRC cell lines (Ma et al., 2018). On the other hand, the ubiquitin specific peptidase 22 (USP22) binds to the promoter of AP4 to activate its transcription to enhance EMT and metastasis formation of CRC cell lines (Li et al., 2017).
In mouse embryonic fibroblasts (MEFs) loss of Ap4 leads to premature senescence through induction of the known target p21 as well as the additional CDK (Cyclin dependent kinase)-inhibitor p16. c-Myc activation is not able to repress p21 and p16 in the absence of Ap4, indicating that Ap4 is necessary for the regulation of proliferation
Introduction
11 by c-Myc (Jackstadt et al., 2013a). Ectopic expression of both Ap4 and Ras (Rat sarcoma protein)combined with loss of p53 leads to cellular transformation of MEFs (Jackstadt et al., 2013a). In addition, AP4 is regulated on posttranscriptional level, since it is a target for proteasome-dependent degradation by the βTrCP ubiquitin ligase (D'Annibale et al., 2014). This degradation takes place in the G2 phase of the cell cycle and is mediated by phosphorylation of a conserved degron. Furthermore, the ectopic expression of a non-degradable stabilized AP4 mutant revealed that βTrCP-dependent degradation of AP4 is required for the fidelity of mitotic division, as it increase chromosomal instability leading to cancer initiating mutations.
These results indicate an important role of AP4 in cancer development and progression. However, the organismal function of AP4 in the intestinal epithelium and its relevance for intestinal tumor formation has so far not been analysed using a genetic approach.
1.8 The homeostasis of the intestinal epithelium
The intestinal epithelium is one of the fastest dividing tissues in the body. The small intestine contains crypts and villi, where each villus is surrounded by at least 6 crypts (Figure 7). In contrast to the small intestine the colon does not contain villi. In the small intestine the crypt base harbors 4-6 intestinal stem cells (ISCs) surrounded by supporting Paneth cells (Barker et al., 2007). Paneth cells are differentiated cells, which are exclusively located in the base of the crypt (Clevers and Bevins, 2013). Lgr5 (Leu-rich repeat-containing G protein-coupled receptor 5)-positive ISCs generate all differentiated cells in the intestine (Barker et al., 2007). The Lgr5+ ISCs divide asymmetrically to self-renew and to generate highly proliferative, Lgr5-negative transit amplifying (TA) cells, which differentiate into absorptive enterocytes or secretory cells, such as goblet, Tuft and entero-endocrine cells (Barker, 2014; Barker et al., 2007; Beumer and Clevers, 2016; Sancho et al., 2004). Goblet cells are located in both the crypt and the villus, and function as mucus producing cells. Paneth cells are derived from Lgr5+ precursors generated by asymmetric division of ISCs, which proliferate slowly and therefore represent label-retaining cells (LRCs) (Buczacki et al., 2013). Paneth cells control the maintenance of the ISCs by secretion of Wnt and Notch ligands, which suppress differentiation into the secretory lineage, and protect ISCs from bacteria and fungi through secretion of Lysozyme and Cryptdin (Clevers and
Introduction
12 Bevins, 2013; Sato et al., 2011b). However, the presence of Paneth cells in the colon is currently unknown. So-called deep crypt secretory (DCS) cells were identified in colonic crypts, which may have Paneth cell like functions (Sasaki et al., 2016). DCS cells are positive for Reg4 (Regenerating Family Member 4) and intermingled with ISCs.
Figure 7 The structural organization and homeostasis of the intestinal epithelium. Left panels: structural organization of the small intestine (a) and colon (b) shown by electron microscopy. Middle panels: Schematic representation of the cellular organization in the small intestine (a) or colon (b). Right panels: Schematic representation of cellular differentiation in the small intestine (a) and colon (b). Taken from (Barker, 2014).
LGR5+stem cell: Leu-rich repeat-containing G protein-coupled receptor 5-expressing stem cell, CBC stem cells: crypt base columnar stem cells, TA cell: transit-amplifying cell.
Introduction
13 The homeostasis of the intestinal epithelium is regulated mainly by the Wnt/β-catenin and Notch pathways, which control ISC maintenance and differentiation (Beumer and Clevers, 2016; Sancho et al., 2004). During the upward migration of cells these signals become weaker and cells differentiate to replace the epithelial cells lost by anoikis (Figure 7) (Clevers and Bevins, 2013). The turnover of epithelial cells occurs every 3-5 days in the small intestine and every 3-5-7 days in the colon, while Paneth cells are long-lived cells surviving 3-6 weeks (Barker, 2014). In addition to the fast cycling ISCs quiescient Bmi1+ (B lymphoma Mo-MLV insertion region 1 homolog) ISCs reside at position +4 above the crypt base (Sangiorgi and Capecchi, 2008). These cells start to proliferate to rebuild the Lgr5+ ISC pool after tissue injury (Tian et al., 2011).
1.9 The role of the Wnt and Notch pathway in intestinal homeostasis
The maintenance of intestinal stem cells is positively controlled by the Wnt, Notch and Egf (epidermal growth factor) pathway as well as negatively controlled by the Bmp (bone morphogenetic protein) pathway (Figure 8) (Sato and Clevers, 2013). Wnt/β -catenin signaling activity is strongest in the intestinal crypt base and decreases as a gradient in the direction towards the villi (Batlle et al., 2002; Munoz et al., 2012; Van der Flier et al., 2007), suggesting an important role at the crypt base. Paneth cells are the main source of Wnt ligands to activate Wnt signaling in ISCs, which is important for the stem cell maintenance and self-renewal capability (Figure 8) (Clevers et al., 2014; Gregorieff and Clevers, 2005; Sato and Clevers, 2013; Sato et al., 2011b). However, also stroma cells provide Wnt signals to the ISCs (Clevers et al., 2014).
Introduction
14 Figure 8 Interaction of intestinal stem cells with their niche. A Schematic representation of the localization of small intestinal crypts. Gradients of expression of Wnt, Bmp, and Egf signals are acting along the crypt axis. B Scheme of the stem cell niche. C Egf, Notch and Wnt signals positively control stem cell characteristics/stemness, whereas Bmp signals negatively control stem cell characteristics of intestinal epithelial cells. Taken from (Sato and Clevers, 2013).
A complete abrogation of Wnt signaling by deletion of Tcf4 results in a complete loss of Lgr5+ ISCs (Korinek et al., 1998; van Es et al., 2012), while inactivation of Ctnnb1/ β-catenin results in loss of ISCs due to increased differentiation and as a consequence
loss of all intestinal crypts (Fevr et al., 2007). However, partial inhibition of Wnt signaling results only in a reduction of ISCs (Huels et al., 2018). Furthermore, overexpression of the Wnt inhibitor Dkk1 (Dickkopf-related protein 1) results in decreased proliferation and loss of intestinal crypts (Kuhnert et al., 2004; Pinto et al., 2003). However, a constitutively active Wnt signaling in intestinal stem cells, but not in progenitors or differentiated cells of mice can effectively initiate colorectal cancer (Barker et al., 2009; Zhu et al., 2009). These results confirm the importance of a well-balanced Wnt/β-catenin signaling in the homeostasis and tumorigenesis in the intestine.
Both the Notch receptor and the Notch target gene Hes1 are expressed in ISCs at the base of intestinal crypts as well as in some cells of the TA unit, suggesting a critical role of active Notch signaling in ISCs and their progenitors (Fre et al., 2005; Jensen et
Introduction
15 al., 2000; Kayahara et al., 2003; Riccio et al., 2008). In the intestinal crypt base Paneth cells present the Notch ligands to the Notch receptors present on ISCs, thereby activating Notch signaling and maintaining the stemness character (Figure 8) (Sato and Clevers, 2013; Sato et al., 2011b). The main ligands mediating the activation of Notch signaling important for the homeostasis of ISCs in the intestine probably are the Dll1 and Dll4 ligands. Mutation of these two ligands reduced proliferation of ISCs and forced them to differentiate, while mutation of the ligand Jag1 had no effect on proliferation and differentiation (Pellegrinet et al., 2011).
Since the Notch1 and Notch2 receptor are expressed in the crypt base of the intestine, while the Notch3 and Notch4 receptor are mostly expressed in the villus, only Notch1 and Notch2 seems to play a role in the maintenance of ISCs (Riccio et al., 2008). Partial Notch inhibition by the γ-secretase inhibitor Dibenzazepine (DBZ) or by deleting Notch1 (but not Notch2) leads to a decrease of ISCs, an increase in secretory cells such as goblet and Paneth cells, a decrease in the expression of the Notch target gene Olfm4 (Olfactomedin 4) as well as reduced stem cell proliferation (Carulli et al., 2015; VanDussen et al., 2012). Notch1 may be the main factor regulating intestinal homeostasis, since conditional deletion of Notch1 leads to the known phenotype of Notch inhibition as well as a decreased expression of the Notch target genes Hes1,
Olfm4 and c-Myc, while no differences can be seen after conditional deletion of Notch2
alone (Carulli et al., 2015). However, a complete loss of Notch signaling, e.g. by simultaneous Notch1 and Notch2 receptor deletion or by Rbp-Jκ (Csl) deletion results in a complete loss of ISCs (Riccio et al., 2008; van Es et al., 2010; van Es et al., 2005b; VanDussen et al., 2012). In addition, complete loss of Notch signaling results in more hyperplasia in goblet and Paneth cells as seen after deletion of Notch1 alone (Carulli et al., 2015), indicating a role of Notch2 in stem cell maintenance and secretory cell differentiation in combination with Notch1. Taken together, these results show that intestinal homeostasis is dependent on a functional Notch signaling pathway.
Aims of the study
16
2. Aims of the study
The present study had the following aims:
• Genetic analysis of Ap4 in an ApcMin mouse model
• Characterization of pathways and mechanisms by which Ap4 influences tumorigenesis and intestinal homeostasis
Materials
17
3. Materials
3.1 Chemicals and reagents
Application Chemical compound Supplier
IHC, IF, Histology
30% H2O2 Carl Roth
Rabbit serum PAA Laboratories
Roti®-Histokitt II Carl Roth
Antibody diluent, background-reducing Dako
Pro taqs IX pH10 BioCyc
Target retrieval solution, citrate pH 6 Dako TRS (Target retrival solution) pH 6 Dako
DAPI Carl Roth
ProLong gold antifade reagent Life Technologies
Hematoxylin Vector Laboratories
Hematoxylin Waldeck
Eosin Sigma-Aldrich
Alcian blue BioOptica
Periodic acid Merck Millipore
Schiff`s reagent Sigma-Aldrich
WB
Rotiphorese gel 30 (37,5:1) Carl Roth Ammonium peroxodisulfate (APS) Carl Roth Tetramethylethylenediamine (TEMED) Carl Roth Sodium dodecyl sulfate (SDS) Carl Roth
β-mercaptoethanol Sigma-Aldrich
Complete mini protease inhibitor cocktail Roche Diagnostics
Bradford reagent Bio-Rad
PageRuler™ plus prestained protein ladder Thermo Fisher Scientific Immobilon-P PVDF,0.45µm membrane Merck Millipore
Skim milk powder Sigma-Aldrich
Methanol Carl Roth
Tween-20 Sigma-Aldrich
Nonidet®P40 substitute Sigma-Aldrich
ECL/HRP substrate Merck Millipore
Western lightning plus ECL Perkin Elmer
Organoids
Growth factor reduced, phenol red-free matrigel Corning
Advanced DMEM/F12 Gibco/Life Technologies
Penicillin-streptomycin Gibco/Life Technologies
Glutamax Gibco/Life Technologies
Hepes Gibco/Life Technologies
Noggin Preprotech
N2 Gibco/Life Technologies
B27 Gibco/Life Technologies
EGF Preprotech
RSPO1 Sinobiological
Y-27632 MedBiochem Express
Wnt-3a Abcam
4-hydroxytamoxifen (4-OHT) Sigma-Aldrich
Materials
18
Application Chemical compound Supplier
ISH
Kaiser glycerine gelantin Merck Millipore
Formamide Sigma-Aldrich
Paraformaldeyd Merck Millipore
3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) Sigma-Aldrich Diethyl pyrocarbonate (DEPC) Sigma-Aldrich
Blocking solution Roche Diagnostics
NBT/BCIP solution, ready to use Sigma-Aldrich
RNA from Yeast Sigma-Aldrich
Heparin sodium Sigma-Aldrich
qChiP
Protein G agarose/salmon sperm DNA Merck Millipore
Fatty acid-free BSA Sigma-Aldrich
37% formaldehyde Merck Millipore
Cell culture
FBS (Fetal Bovine Serum) Invitrogen
Penicillin-streptomycin Gibco/Life Technologies DMEM–Dulbecco's Modified Eagle Media Gibco/Life Technologies McCoy's 5A (Modified) media Gibco/Life Technologies RPMI 1640 media without L-glutamine Gibco/Life Technologies Hank's Balanced Salt Solution (HBSS) Gibco/Life Technologies DMSO (Dimethyl Sulfoxide) Carl Roth
FuGENE® HD transfection reagent Promega Corporation HiPerFect transfectin reagent QIAGEN
Lipofectamine® 2000 transfection reagent Invitrogen
Opti-MEMTM reduced serum media Gibco/Life Technologies
Doxycycline (DOX) Sigma-Aldrich
Puromycin dihydrochloride Sigma-Aldrich Cloning /
generation of vectors
Ampicillin Carl Roth
LB agar (Lennox) Carl Roth
LB medium (Luria/Miller) Carl Roth
Universal agarose PeqGold PeqLab
Ethidium bromide Carl Roth
Gene ruler 1kb DNA ladder Thermo Fisher Scientific
PCR, Gel electro-phoresis, q-PCR
Deoxynucleotides (dNTPs) Thermo Fisher Scientific Gene ruler low range DNA laddder Thermo Fisher Scientific Gene ruler 100bp plus DNA laddder Thermo Fisher Scientific Gene ruler 1kb DNA ladder Thermo Fisher Scientific
Universal agarose PeqGold PeqLab
Ethidium bromide Carl Roth
Fast SYBR® green master mix Applied Biosystems
Trizol Invitrogen
Luciferase reporter assays
Ampicillin Sigma-Aldrich
LB-Agar (Lennox) Carl Roth
LB-Medium (Luria/Miller) Carl Roth
Hi-Di™ Formamide Applied Biosystems
Universal agarose PeqGold PeqLab
Gene Ruler 1kb DNA ladder Thermo Fisher Scientific
Ethidium bromide Carl Roth
FuGENE® HD transfection reagent Promega Corporation Opti-MEM® Reduced Serum Medium Gibco/Life Technologies Electron
microscopy
Osmium tetroxide Serva Electrophoresis
Materials
19
Application Chemical compound Supplier
DBZ-treatment
Tween-80 Sigma-Aldrich
Methocel® MC low viscosity Sigma-Aldrich
Dibenzazepine (DBZ) Axon Medchem
3.2 Enzymes
Application Enzyme Supplier
Organoids Collagenase Type IV Merck Millipore
Dispase Type II Sigma-Aldrich
ISH Proteinase K Sigma-Aldrich
Cell culture Trypsin-EDTA (0.5%, 10x, phenol-red free) Gibco/Life Technologies Generation
of vectors
restriction endonucleases New England Biolabs
T4 DNA ligase Thermo Fisher Scientific
qPCR DNase I (RNase-free) Sigma-Aldrich
PCR FIREPol® DNA Polymerase Solis BioDyne
Hot FIREPol® DNA Polymerase Solis BioDyne
3.3 Kits
Application Kit Supplier
IHC
DAB Substrate Chromogen System Vector laboratories DAB+ Substrate Chromogen System Dako
Vectastain Elite ABC HRP detection Kit Vector laboratories AEC Substrate Chromogen System Thermo Fisher Scientific Immpress HRP Anti Goat Ig Vector laboratories Immpress HRP Anti Rabbit Ig Vector laboratories Impress Excel Staining Kit, Anto Rabbit-Ig Vector laboratories Immpress HRP Anti Mouse Ig Vector laboratories M.O.M. (mouse on mouse) Kit Vector laboratories
WB BCA Protein Assay Kit Thermo Fisher Scientific
ISH
DIG Northern Starter Kit RocheDiagnostics BCIP/NBT substrate system Sigma Aldrich Cloning /
generation of vectors
QIAquick Gel Extraction Kit QIAGEN
Pure Yield™ Plasmid Midiprep System Promega Corporation
QIAprep Spin Miniprep Kit QIAGEN
BigDye Terminator v1.1 cycle sequencing Kit Applied Biosystems
Dye Ex 2.0 Spin Kit QIAGEN
qPCR
High Pure RNA Isolation Kit RocheDiagnostics
RNeasy Kit QIAGEN
Verso cDNA Synthesis Kit Thermo Fisher Scientific Luciferase
reporter assays
Materials
20
3.4 Antibodies
3.4.1 Primary antibodies
epitope source company catalog
no. use
dilution mouse
dilution human
α-Tubulin mouse Sigma-Aldrich #T9026 WB 1:1000 1:1000
β-catenin mouse BD
Pharmingen 610154 IHC 1:300 BrdU rat AbD Serotec MCA2060 IHC 1:400 Cleaved
caspase-3 rabbit Cell signaling #9661 IHC 1:100 c-myc rabbit Merck
Millipore #06-340 IHC 1:300 GFP rabbit Santa Cruz SC-8334 IHC; IF 1:700; 1:700
Hes1 rabbit Cell signaling #11988 IHC; WB 1:50; 1:1000 1:50; 1:1000
Ki67 rat Dako E0468 IHC 1:500
Lysozyme rabbit Biozol, Lifespan Biosciences LS- C138144-100 IHC; IF 1:4000; 1:4000
NICD1 rabbit Cell Signaling #4147 IHC; WB 1:50; 1:1000 1:100; 1:1000 TFAP4 mouse AbD Serotec MCA4993Z IHC; IF;
WB; ChiP 1:100; 1:100; 1:1000; 3µg 1:400; 1:1000 VSV rabbit Sigma-Aldrich #4888 WB 1:7500
Rabbit IgG Biozol
Diagnostica BZL04060 IHC; IF Rabbit (DA1E)
mAB IgG Cell Signaling 3900 IHC
Mouse IgG2a Biozol
Diagnostica
SER-
MCA929-100
IHC; IF
Mouse IgG1 Southern
Biotech
SBA-0102-01 IHC
Rat IgG2a Biozol
Diagnostica BZL01284 IHC Mouse IgG Sigma-Aldrich M7023 ChiP
ChiP: Chromatin Immunoprecipitation; IF: Immunoflourescence; IHC: Immunohistochemistry; WB: Western Blotting;
Materials
21
3.4.2 Secondary antibodies
name source company catalog no. use dilution Used for pAB Anti-mouse-HRP goat Promega Corporation #4021 WB 1:10000 α-Tubulin, TFAP4
Anti-Rabbit-HRP goat Sigma-Aldrich #A0545 WB 1:10000
c-Myc, NICD1, Hes1
Anti-mouse-Alexa 555 goat Invitrogen A-21422 IF 1:500 TFAP4 Anti-Rabbit-FITC donkey Jackson Immuno Research 711-095-152 IF 1:500 GFP, Lysozyme
Anti-Rat-biotinylated rabbit Dako E0468 IHC 1:500 Ki67
ChiP: Chromatin Immunoprecipitation; IF: Immunoflourescence; IHC: Immunohistochemistry; WB: Western Blotting;
Materials
22
3.5 Buffers and solutions
Immunohistochemistry:
10x PBS (1l):
80g NaCl; 1 g KCl; 14.42 g Na2HPO4*2H2O; 2 g KH2PO4; ad 1 l ddH2O
Tris-Buffer:
43.90 g NaCl; 34.25 g Tris-HCL; 4.50 g Tris-Base; add ddH2O to 5 l
Western Blotting:
2x Laemmli buffer:
125 mM Tris/HCl (pH 6.8); 4% SDS; 20% glycerol; 0.05% bromophenol blue (in H2O); 10% β
-mercaptoethanol (added right before use)
RIPA lysis buffer (for protein lysates):
1% NP40 (Nonidet P-40); 0.5% sodium deoxycholate; 0.1% SDS; 150 mM NaCl; 50 mM Tris/HCl (pH 8.0), add 1 tablet of protease (Roche Diagnostics) to 10 ml of RIPA buffer
10x Tris-glycine-SDS running buffer (5l, for SDS-PAGE): 720 g Glycin; 150 g Tris; 50 g SDS; pH 8.3-8.7; ad 5 l ddH2O
Towbin buffer (for Western blotting):
200 mM glycine; 20% methanol; 25 mM Tris (pH 8.6)
10x TBS-T (5l):
500 ml 1M Tris (pH 8.0); 438.3 g NaCl; 50 ml Tween20; ad 5 l ddH2O
Genotyping of mice:
10x Gitschier`s Buffer (10x GB):
Materials 23 Soriano Buffer: 5040 µl of ddH2O; 600 µl of 10x GB; 300 µl of 10% Triton X-100; 60 µl 2-Mercaptoethanol 10x Vogelstein PCR buffer: 166 mM (NH4)2SO4; 670 mM Tris/HCL (pH 8.8); 67 mM MgCl2; 100 mM β-mercaptoethanol In situ Hybridization
Acetic anhydride solution:
0.2 % acetic anhydride in 0.1 M trietanolamine (pH 8.0)
Tris/NaCl Buffer:
0.1 M Tris/HCl (pH 7.5); 0.15 M NaCl; 0.1% Tween20
Blocking Solution:
1 % blocking powder (Roche Diagnostics) in 1x Tris/NaCl buffer (heat to 65°C to dissolve)
DEPC-H2O:
1 ml Diethyl-Pyrokarbonat (DEPC) (Sigma) in 1 L H2O (autoclave to inactivate DEPC)
Hybridization solution:
50% formamide, 5x SSC (pH 4.5); 2% Blocking Powder (Roche Diagnostics); 0.05% CHAPS (Sigma); 5 mM EDTA; 50 µg/ml heparin; 1 µg/ml yeast RNA (heat to 65°C to dissolve)
NBT/BCIP Solution:
10 mg of NBT (Sigma) in 1ml of DEPC-H2O
NTM Buffer:
0.1 M Tris/HCl (pH 9.5); 0.1 M NaCl, 0.05 M MgCl2
PFA:
4% Paraformaldedyde (PFA) in PBS (heat to 65°C and add NaOH to dissolve)
1x PBS
Materials
24 SSC, 20X:
175.3g NaCl; 88.2g sodium citrate*2H2O; add DEPC-H2O to 1l (adjust pH to 7.5 or 4.5)
qChiP:
TE buffer:
10 mM Tris/HCL (pH 7.5); 1 mM EDTA
Glycine buffer: 1 M glycine in ddH2O
Triton X-100 dilution buffer:
100 mM Tris/HCl (pH 8.6); 100 mM NaCl; 5 mM EDTA (pH 8.0); 0.2 % NaN3; 5 % Triton X-100
SDS buffer:
50 mM Tris (pH 8.1); 0.5% SDS; 100 mM NaCl; 5 mM EDTA
Immunoprecipitation (IP) buffer:
Mix 1 part of Triton X-100 dilution buffer and 2 parts of SDS buffer
LiCl/detergent wash:
0,5 % deoxycholic acid (sodium salt); 1 mM EDTA; 250 mM LiCl; 0.5 % NP-40; 10 mM Tris/HCl (pH 8.0); 0.2 % NaN3
Buffer 500:
0.1 % deoxycholic acid; 1 mM EDTA; 50 mM HEPES (pH 7.5); 500 mM NaCl; 1 % Triton X-100; 0.2 % NaN3
Mixed micelle buffer:
150 mM NaCl; 20 mM Tris/HCl (pH 8.1); 5 mM EDTA (pH 8.0); 5.2 % Sucrose; 0.02 % NaN3,
Materials 25 Elution buffer: 10 mM EDTA, 1 % SDS; 50 Mm Tris/HCl (pH 8.0) 1x TBS: 1.5 M NaCl; 0.1 M Tris/HCl (pH 7.4) 1x PBS: 13.7 mM NaCl; 2.7 mM KCl; 80.9 mM Na2HPO4; 1.5 mM KH2PO4 (pH 7.4)
Materials
26
3.6 Oligonucleotides
3.6.1 Oligonucleotides used for genotyping
Name Sequence (5`-3`)
Ap4 Primer a Ap4 Primer b Ap4 Primer c ApcMin wt ApcMin com ApcMin mut Vil-Cre Fwd Vil-Cre Rev Lgr5 com Lgr5 wt rev Lgr5 mut rev Cmv-Cre trans1 Cmv-Cre trans2 Cmv Int Con Fwd Cmv Int Con Rev
GCCTAAGAGTAGGTGCTCTGC GCGAGCAAATGAACTGTTGAC CGTACGCCGGCTTAAGTGTA GCCATCCCTTCACGTTAG TTCCACTTTGGCATAAGGC TTCTGAGAAAGACAGAAGTTA CGCGAACATCTTCAGGTTCT CAAGCCTGGCTCGACGGCC CTGCTCTCTGCTCCCAGTCT ATACCCCATCCCTTTTGAGC GAACTTCAGGGTCAGCTTGC GCGGTCTGGCAGTAAAAACTATC GTGAAACAGCATTGCTGTCACTT CTAGGCCACAGAATTGAAAGATCT GTAGGTGGAAATTCTAGCATCATCC
3.6.2 Oligonucleotides used for qPCR
Name Sequence (5`-3`)
mouse B2m Fwd CCGGCCTGTATGCTATCC
mouse B2m Rev CTTGCTGAAGGACATATCTGACA mouse Ap4 Fwd
mouse Ap4 Rev mouse Spdef Fwd mouse Spdef Rev mouse EpCam Fwd mouse EpCam Rev
mouse Lysozyme Fwd mouse Lysozyme Rev mouse Cryptdin Fwd
mouse Cryptdin Rev mouse Gob5 Fwd mouse Gob5 Rev mouse Muc2 Fwd mouse Muc2 Rev mouse Smoc2 Fwd mouse Smoc2 Rev mouse Lgr5 Fwd mouse Lgr5 Rev mouse Olfm4 Fwd mouse Olfm4 Rev
mouse Cdkn1a (p21) Fwd mouse Cdkn1a (p21) Rev mouse Ctnnb1 Fwd
mouse Ctnnb1 Rev mouse Sox4 Fwd mouse Sox4 Rev mouse Axin2 Fwd mouse Axin2 Rev
TCAAGCGCTTTATCCAGGAG CAATGCCCTCATCCTTGTCT AACATGTATCCCGACGATAGCAGC TCAATATCTTTCAGGACCTCGCCC TTGCTCCAAACTGGCGTCTA ACGTGATCTCCGTGTCCTTGT ATGGAATGGCTGGCTACTTATGGAG CTCACCACCCTCTTTGCACATTG AGGAGCAGCCAGGAGAAG ATGTTCAGCGACAGCAGAG TGAAATTGTGCTGCTGACCGATGG TGCTGCGAAAGCATCAACAAGACC TGTGGGACTTTTGCCATGTACT GCAAGAGCACCTGTGATCCA GAAGAAGATATTGCCTCACG TTCCTCAAGAGCTGACTGAT GAGGAAGCGCTACAGAATTTGAGA GTGGCACGTAGCTGATGTGG TGGCCCTTGGAAGCTGTAGT ACCTCCTTGGCCATAGCGAA AACATCTCAGGGCCGAAA TGCGCTTGGAGTGATAGAAA TGCTGAAGGTGCTGTCTGTC AGTCGCTGCATCTGAAAGGT GCTGCATCGTTCTCTCCAGA AAACAGGTAGACGCGCTTCA ATGCTAGGCGGAATGAAGATG GGAGACAACGCTGTTGTTCTC
Materials
27 mouse Ascl2 Fwd
mouse Ascl2 Rev mouse Dll1 Fwd mouse Dll1 Rev mouse Notch1 Fwd mouse Notch1 Rev mouse Hes1 Fwd mouse Hes1 Rev mouse Jag1 Fwd mouse Jag1 Rev mouse Jag2 Fwd mouse Jag2 Rev mouse Dll4 Fwd mouse Dll4 Rev mouse Hey1 Fwd mouse Hey1 Rev
mouse Tcf7 (Tcf1) Fwd mouse Tcf7 (Tcf1) Rev mouse c-Myc Fwd
mouse c-Myc Rev mouse EphB3 Fwd mouse EphB3 Rev human β-ACTIN Fwd
human β-ACTIN Rev
human AP4 Fwd human AP4 Rev human c-MYC Fwd human c-MYC Rev human NOTCH1 Fwd human NOTCH1 Rev human NRARP_Fwd human NRARP_Rev human HES1 Fwd human HES1 Rev
GCCCGTGAAGGTGCAAAC ACAGGAAAAGTGCTCGCGA CATGAACAACCTAGCCAATTGC GCCCCAATGATGCTAACAGAA GCAGATGCTCAGGGTGTCTT GCCAGGATCAGTGGAGTTGT TCAGCGAGTGCATGAACG TGCGCACCTCGGTGTTAAC TCTCTGACCCCTGCCATAAC TTGAATCCATTCACCAGATCC GGCAACTCCTTCTACCTGCC GTCATTGTCCCAGTCCCAGG CCCTCACCTGGATTACCTAC GAATCTGCTTGTTAGGGATG TGAGCTGAGAAGGCTGGTAC ACCCCAAACTCCGATAGTCC TGCAGCTATACCCAGGCTGG CCTCGACCGCCTCTTCTTC TGACCTAACTCGAGGAGGAGCTGGAATC AAGTTTGAGGCAGTTAAAATTATGGCTGAAGC AAGAGACTCTCATGGACACGAAAT ACTTCCCGCCGCCAGATG TGACATTAAGGAGAAGCTGTGCTAC GAGTTGAAGGTAGTTTCGTGGATG GCAGGCAATCCAGCACAT GGAGGCGGTGTCAGAGGT GCTGCTTAGACGCTGCTGGATTT TAACGTTGAGGGGCATCG TGATGAGGTCCTCCAGCAT TGATGAGGTCCTCCAGCAT TTCGAACCCGAAATCCTG GCCACAGAAACCAGGAAGG GAAGCACCTCCGGAACCT GTCACCTCGTTCATGCACTC
3.6.3 Oligonucleotides used for qCHIP
Name Sequence (5`-3`)
mouse Sox4 (A) Fwd TTCATGGGCCGCTTGATGT mouse Sox4 (A) Rev CAACAACGCGGAGAACACTG mouse Sox4 (B) Fwd CGCGTGCAATGAGAAGCTC mouse Sox4 (B) Fwd CACACACACAGAGGCAAACG mouse Ascl (A) Fwd GCAGAGGTCAGTCAGCACTT mouse Ascl (A) Rev TTCTTCACAGCTGCATCCCT mouse Ascl (B1) Fwd ACAAACAAACGCCGGTCTTG mouse Ascl (B1) Rev GCCTGACACTTAGCGCCA mouse Ascl (B2) Fwd CTCCATCGGGCTTAGCTCTC mouse Ascl (B2) Rev TCTCTGTCCTGCGCCTCTAC mouse Ascl (C1) Fwd GCGTGGCTCCAGAGATGG mouse Ascl (C1) Rev TTCTCACACTCAAGGGGCAC mouse Ascl (C2) Fwd GTGCCCCTTGAGTGTGAGAA mouse Ascl (C2) Rev GCATGAGGGGCTAAATGGGT mouse Tcf7 (A) Fwd TCTTTGGGTAGAAGGCAGCC
Materials
28 mouse Tcf7 (A) Rev GCCTCAGCCAAAGTCATTCTG
mouse Tcf7 (B) Fwd AATCAGCCATCACCACCACC mouse Tcf7 (B) Rev TGAGGGCAGAGGAGGAAGAA mouse Notch1 (A) Fwd ACACTCAGCTCCCCGGAT mouse Notch1 (A) Rev ACAATGGGCCGCTCTGATTC mouse Notch1 (B) Fwd CCTACCTCTTGCGGCGAG mouse Notch1 (B) Rev CCGGTGGTGTGCGTCAAC mouse Dll1 (A) Fwd GTTTGGTGTGTGTCGTTCGC mouse Dll1 (A) Rev AGCTCTTTCTCTCCGCATTGT mouse Dll1 (B) Fwd ACATGAGAAAAGGGGAGGCG mouse Dll1 (B) Rev AGGAAGGAGAGGCATAGGGG mouse Jag1 (A) Fwd CTCGCGCTCCCCTTCTTTTA mouse Jag1 (A) Rev CATTGTGTTACCTGCAGCCG mouse Jag1 (B) Fwd CTTGCAAGCCCCAGGTGTAG mouse Jag1 (B) Rev CTCTGGGCTCGCTTGCTG mouse Jag2 (A) Fwd CAAGAGCACGCGCCCCAGG mouse Jag2 (A) Rev CTTTCAGTTCGCCTGGCCGGTAC mouse Jag2 (B) Fwd CTCAGCAGCTCCCCGTTC
mouse Jag2 (B) Rev CACTCTGCGCTGCCTTATTT mouse Jag2 (C) Fwd GGGACGAGACTGACAGCTC mouse Jag2 (C) Rev CTCGCCTCCTTTAAAGCTCG mouse Hes1 Fwd CACACACCCCACACGCAG mouse Hes1 Rev CCAAGAAGGTAAATAGCAGCTG mouse Dll4 (A) Fwd TGTACTCCCTCACTAGCCCG mouse Dll4 (A) Rev GTAATCCAGGTGAGGGCGAC mouse Dll4 (B) Fwd CTCCTTCTCTCGGTCCCTGT mouse Dll4 (B) Rev GCAGATGCGGAAGAAAGTCC mouse Dll4 (C) Fwd GGGACAAGAATAGCGGCAGT mouse Dll4 (C) Rev GAAAGGAGCTCTGGTGTCCC mouse Cdkn1a (A1) Fwd AATTGAAGAGGTGGGGCTGC mouse Cdkn1a (A1) Rev TCTGGGGTCTCTGTCTCCAT mouse Cdkn1a (A2) Fwd TCCCACTTTGCCAGCAGAAT mouse Cdkn1a (A2) Rev CCAGGCACACACACACAGAT mouse Cdkn1a (B) Fwd CCCAGAAGTGTGTGTGTGTG mouse Cdkn1a (B) Rev ACACCCGTCATCCACCTG mouse Cdkn1a (C) Fwd CCCCAGACGCTTCATCTCTT mouse Cdkn1a (C) Rev TACCACACACATACACACGC mouse EphB3 Fwd AAGAGCGGCCAACTGAACTC mouse EphB3 Rev CTGCCCGTCAACACTCAGG mouse AchR Fwd
mouse AchR Rev
AGTGCCCCCTGCTGTCAGT CCCTTTCCTGGTGCCAAGA
Materials
29
3.7 siRNAs
The following siRNAs were purchased from Ambion:
AP4-specific siRNA: 5‘-GUGAUAGGAGGGCUCUGUAG-3‘
Silencer negative control siRNA: 5-UUGUCUUGCAUUCGACUAAUU-3 .
3.8 Vectors
Name Insert Source
Bluescript II plasmid
p695-pBS-mOlfm4 Mouse Olfm4 ORF Prof. Dr. Hans Clevers pCMV6-Entry-Lgr5 Mouse Lgr5 ORF Origene, cat.no. MR219702 pCMV6-Entry-Smoc2 Mouse Smoc2 ORF Origene, cat.no. MR207121 pBluescriptII-KS empty PromegaCorporation
pRTR empty (Jackstadt et al., 2013c)
pRTR-Ap4-VSV AP4-VSV ORF (Jackstadt et al., 2013c) pGA-RBPJ Human RBPJ-ORF (Oswald et al., 2001)
pGA empty (Oswald et al., 2001)
ORF: Open Reading Frame
3.9 Mice
Genotype Background Source
Ap4fl/fl
C57BL/6
Our lab (Jackstadt et al., 2013a)
ApcMin Dr. Marlon Schneider (LMU)
Lgr5-CreERT2 Prof. Dr. Hans Clevers (Hubrecht
Institute)
Vil-Cre
Prof. Dr. Klaus-Peter Jannsen (TUM)
Vil-CreERT2 CMV-Cre
The Jackson Laboratory
Flp
Materials
30
3.10 Cell lines
Species Cell lines Medium Supplier
Human CRC cell lines
SW620 DMEM Medium + 10% FBS + 1% P/S -- -- DLD-1 McCoy`s 5A Medium + 10% FBS + 1% P/S -- HCT15 -- Colo320
RPMI1640 w/o glutamax + 1% Glutamax +
10% FBS + 1% P/S
--
Murine CRC cell line CT26
RPMI1640 w/o glutamax + 1% Glutamax + 10% FBS + 1% P/S
ATCC
Human kidney cell line HEK293 DMEM Medium +
10% FBS + 1% P/S DSMZ ATCC: The Global Bioresource Center; https://www.atcc.org/
DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; https://www.dsmz.de/
3.11 Software
application software Supplier
Data analysis and figure generation
Excel Microsoft Cooperation
Prism5 Graphpad Software Inc.
WB
Varioskan Flash Multimode Reader Thermo Scientific KODAK MI SE software Carestream Health
qPCR ND1000 V3 PEQLAB
LightCycler 480 Roche Diagnostics
Cell Culture cFlow Software Accuri
Sequencing analysis DNA Sequencing Analysis Software v5 Applied Biosystems
BioEdit BioEdit
IF ZEN 2009 Carl Zeiss
IHC, ISH,
tumor counting Axiovision Carl Zeiss
Organoids Nikon NIS-Elements Nikon
Luciferase reporter
Materials
31
3.12 Laboratory equipment
application Device Supplier
qPCR ND 1000 NanoDrop Spectrophotometer PEQLAB
LightCycler480 Roche Diagnostics
WB
Mini-PROTEAN®-electrophoresis system Bio-Rad HTU SONI130
G. Heinemann Ultraschall- und Labortechnik Varioskan Flash Multimode Reader Thermo Scientific Mini Trans-Blot® Electrophoretic Transfer Cell Bio-Rad
Powerpac 300 Power Supply Bio-Rad
biophotometer plus eppendorf
EPS 600 power supply Pharmacia Biotech
440CF imaging system Eastman Kodak
Cell culture
Herasafe KS class II safety cabinet Thermo Fisher Scientific Neubauer counting chamber Carl Roth BD AccuriTM C6 Flow Cytometer Instrument BD Accuri Axiovert 25 microscope equipped with an
Axiocam 105 color camera Carl Zeiss
Sequencing ABI 3130 genetic analyzer capillary sequencer Applied Biosystems
IF LSM700 confocal microscope Carl Zeiss
IHC, ISH Axioplan 2 imaging microscope
AxioCam HRc camera Carl Zeiss
Electron microscopy Libra 120 transmission electron microscope Carl Zeiss Organoids
Stemi 2000-C stereo microscope Carl Zeiss Nikon AZ-100 macroscope equipped with a
Nikon DS-Fi3 color camera equipped with a 5.9 megapixel CMOS image sensor.
Nikon
Tumor counting
Stemi 2000-C stereo microscope Carl Zeiss Nikon D5100 digital camera equipped with a
Nikon AF-S Nikkor 18-55mm 1:3.5-5.6G objective
Nikon Luciferase reporter
assays Orion II Luminometer
Berthold Technologies
Methods
32
4. Methods
4.1 Generation and husbandry of mice
Ap4fl/fl mice and germ-line Ap4 knock-out mice were generated by the Hermeking lab
as follows: targeted embryonic stem cells (ES-cells) with C57BL/6N background were obtained by homologous recombination with a vector containing the Ap4 exons 2-4 flanked by loxP (locus of X-over P1) sites and an intronic neomycin resistance (Neo) cassette flanked by frt (flp recognition target) sites (scheme in (Jackstadt et al., 2013a)). Ap4+/fl mice were generated by injection of targeted ES cells into C57BL/6N
blastocyst. The Neo cassette was removed by crossing to flp (Flippase)-mice (Gronostajski and Sadowski, 1985) and germ-line Ap4 knock-out mice were generated by crossing with CMV(cytomegalovirus)-Cre+/- mice (Schwenk et al., 1995).
Ap4-/- mice showed no overt phenotype and were born at normal Mendelian ratio.
Oligonucleotides used for genotyping are listed in Table 3.6.1. For analysis of the effect of Ap4 inactivation on the ISC number we used Lgr5-eGFP-Cre-ERT2+/- mice (Barker and Clevers, 2007) (obtained from Hans Clevers, University Medical Center Utrecht, The Netherlands) and for specific deletion of Ap4 in intestinal epithelial cells or derived organoids we used Villin-Cre+/- or Villin-Cre-ERT2+/- mice (el Marjou et al., 2004) (obtained from Klaus-Peter Janssen, Technical University Munich, Germany), respectively. ApcMin/+ mice (Moser et al., 1990; Su et al., 1992) (obtained from Marlon
Schneider, Ludwig-Maximilians-Universität, München, Germany) were used to analyze the role of Ap4 in intestinal adenoma development. Mice were kept in individually ventilated cages (IVC) with a 12-hour light/dark cycle and ad libitum access to water and standard rodent diet. For determination of proliferation rates 75 mg/kg BrdU (Bromdesoxyuridin) (Amersham) in PBS (phosphate-buffered saline) was i.p.
(intraperitoneal) injected 1.5 hours before mice were sacrificed. All animal experimentations and analyses were approved by the Government of Upper Bavaria, Germany (AZ 55.2-1-54-2532-4-2014).
4.2 Tissue preparation and adenoma counting
After isolation of intestinal tissue, the colon and small intestine were separated and flushed with PBS to remove stool. The small intestine was dissected into duodenum, jejenum and ileum. The colon and small intestine were opened longitudinally and rolled
Methods
33 with the mucosa oriented outwards and fixed in formalin, dehydrated and embedded into paraffin. For evaluation of tumor numbers each part of the intestine was cut longitudinally and spread on Whatman 3 MM paper. After fixation in formalin adenomas were counted under a Stemi 2000-C stereo (dissection) microscope (Carl Zeiss) with 10x magnification. Pictures of intestines were taken with a Nikon D5100 digital camera with a Nikon AF-S Nikkor 18-55mm 1:3.5-5.6G objective.
4.3 HE and PAS/Alcian blue staining
Formalin-fixed, paraffin-embedded (FFPE) tissue was cut into 2 µm sections on a Microm HM355S rotating microtome (Thermo Fisher Scientific). For hematoxylin and eosin (HE)-staining the slides were de-paraffinized and stained with hematoxylin (Waldeck) for 6 minutes followed by eosin (Sigma-Aldrich) for 2.5 minutes in an automated slide staining device (Tissue-Tek, Prisma). Periodic acid Schiff (PAS)-staining was done by applying Alcian Blue pH 1 (Bio Optica) for 10 minutes followed by periodic acid (Merck Millipore) for 5 minutes, Schiff`s reagent (Sigma Aldrich) for 5 minutes and counterstaining with hematoxylin (Waldeck).
4.4 Immunohistochemistry
FFPE tissue was cut into 2 µm sections on a microtome and de-paraffinized. After antigen retrieval slides were incubated with primary antibody (the primary antibodies used are listed in Table 3.4.1) for 1 hour at room temperature and washed with Tris-HCL (Tris hydrochloride) buffer (pH 7.5) followed by a secondary antibody. Antibodies were detected with the Vectastain Elite ABC (avidin-biotin complex) kit (Vector) using DAB (3,3'-diaminobenzidine) (Vector Laboratories and Dako) for brown stainings or AEC (3-Amino-9-ethylcarbazole) (Thermo Fisher Scientific) for red stainings. The slides were counterstained with hematoxylin (Vector Laboratories) and mounted with Roti®-Histokitt II (Carl Roth). All stainings were performed with the respective IgG (Immunglobulin G) control (Table 3.4.1) as a negative control and without primary antibody as a system control. Images were captured on an Axioplan2 imaging microscope (Carl Zeiss) equipped with an AxioCamHRc Camera (Carl Zeiss). For analysis of cleaved Caspase-3 the AxioVision Software (Carl Zeiss) was used to measure the area for each tumor in mm2.
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34
4.5 In situ hybridization
For detection of ISCs with an Olfm4 mRNA probe, the Bluescript II plasmid p695-pBS-mOlfm4 (kindly provided by Prof. Hans Clevers) was linearized by using a NotI restriction enzyme (New England Biolabs). The pCMV6 entry plasmids containing the
Lgr5 or Smoc2 open reading frame (ORF) were obtained from Origene with the catalog
number MR219702 or MR207121, respectively. Both ORFs were cloned into the pBSII KS plasmid (Promega Corporation) by using the restriction enzymes NotI and KpnI. The pBSII KS-mSmoc2 was linearized with the KpnI restriction enzyme and the pBSII KS-mLgr5 was linearized with the BclI restriction enzyme. The Olfm4, Lgr5 and Smoc2 (SPARC-related modular calcium-binding protein 2) RNA probe was generated by an
in vitro transcription reaction with a RNA-T7 Polymerase by using the DIG Northern
Starter Kit (Roche Diagnostics). During the transcription reaction the probe was labeled with digoxigenin (DIG). The in situ hybridization was performed on freshly prepared 8 µm paraffin sections as described (Gregorieff and Clevers, 2010).
4.6 Isolation of IECs
Each part of the intestine was dissected longitudinally and cut into small pieces. IECs were isolated by shaking the tissue in Hanks´balanced salt solution (HBSS)/ethylene-diamine-tetra-acetic acid (EDTA) at 37◦C for 10 minutes. The supernatant including
IECs was centrifuged and the pellet was washed with ice-cold PBS, frozen in liquid nitrogen and stored at -80◦C until RNA isolation.
4.7 Crypt isolation and organoid culture
Crypt isolation and organoid culture was performed as described before (Sato et al., 2009). The small intestine was opened longitudinally, and the villi were scraped off under a dissection microscope by using a surgical blade. The intestine was cut into small pieces and incubated in 8 mM EDTA in HBSS for 5 minutes to remove the rest of the villi followed by an additional incubation in EDTA for 30 minutes at 4◦C. Isolated
crypts were washed in advanced DMEM (Dulbecco's Modified Eagle's Medium)/F12 (Gibco / Life Technologies) containing Glutamax (Gibco / Life Technologies) and Hepes (Gibco / Life Technologies), passed through a 100 µm cell strainer and either frozen in liquid nitrogen (IEC) or counted and pelleted for culturing (organoids). 200 crypts were mixed with 50 µl of growth factor reduced, phenol red-free matrigel. After
Methods
35 polymerization of the Matrigel 600 µl of crypt culture medium was added. Crypt culture medium consists of advanced DMEM/F12 supplemented with 1:100 P/S (Penicillin/Streptomycin) (Gibco / Life Technologies), 1:100 Hepes, 1:100 Glutamax and the following growth factors: 100 ng/ml Noggin (Preprotech), 1:100 N2 (Gibco / Life Technologies), 1:50 B27 retinoic acid free (Gibco / Life Technologies), 50 ng/ml epidermal growth factor (EGF) (Preprotech), 500 ng/ml R-spondin-1 (RSPO1) (Sinobiological), 10 µM Y-27632 (MedBiochem Express) was added only the first 2 days after isolation/culturing or passaging to avoid anoikis and 100 ng/ml Wnt-3a (Abcam) was added only the first two days after isolation/culturing. Crypt culture medium was changed every 2 days. Organoids were passaged at a 1:6 ratio once a week. For passaging organoids were removed from Matrigel and dissociated mechanically into single-crypt domains before they were transferred into fresh Matrigel. 4-OHT (4-hydroxytamoxifen) (Sigma-Aldrich), diluted in ethanol, was added to the crypt culture medium to a final concentration of 500 nM for at least 12 hours. RNA was isolated using Trizol (Invitrogen) and the RNeasy Mini Kit (QIAGEN).
For generation of tumoroids intestinal adenoma cells from 3 tumors for each ApcMin/+
mouse were isolated by lysis in DMEM containing 4000 units Collagenase Type IV (Merck Millipore) and 125 µg/ml Dispase Type II (Sigma-Aldrich). Single cells were embedded in Matrigel and seeded in 24-well plates (15,000 single cells per 50 µl Matrigel per well). The tumor organoid culture medium was formulated as described before (Sato et al., 2011a). Crypt culture medium (Advanced DMEM/F12 supplemented with 1:100 Penicillin/Streptomycin, 1:100 Hepes and 1:100 Glutamax and growth factors (1:100 N2, 1:50 B27, 50 ng/ml EGF)). 10 µM Y-27632 was added only the first 2 days after isolation/culturing or passaging to avoid anoikis. Counting of the number of organoids per well (6 wells per mouse) was performed 6 days after isolation before the first passaging. Passaging was performed as described above for normal organoids. RNA was isolated using Trizol and the RNeasy Mini Kit. For Western blot analysis tumor organoids were lysed in RIPA lysis buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, Complete Mini protease inhibitors (Roche Diagnostics)).
For tumoroid formation after acute loss of Apc or Apc and Ap4 crypts were isolated as described above. For each well in a 6-well plate approximately 8 drops of 25µl matrigel and 50 crypts each were plated and overlaid with ENR media (containing EGF,
